The structure of the ribosome represents a landmark in the study of translation. However, much less is known about the dynamics of the ribosome during protein synthesis. This proposal investigates the rates and natures of ribosomal conformational changes during various steps in translation. We have developed single-molecule fluorescence spectroscopic methods that allow these measurements. We have shown that ribosomes can be specifically attached to quartz surfaces, and that these ribosomes are active. The specific aims of the proposal will look at critical functional steps in translation. In general, either ribosomal elements (proteins or RNA) will be labeled with fluorescent dyes, or the ligands of the ribosomes (tRNAs or factors) will be labeled. Specific Aim 1 will investigate the basic aspects of ribosome attachment to the surface, stability of ribosomal particles and general fluorescent dye labeling schemes. The peptidyl transferase reaction, and its dependence on various solution conditions will be measured. In Specific Aim 2, the process of tRNA selection will be monitored. In particular, we are interested in the conformational signaling that occurs between the codon-anticodon interaction on the small subunit and the factor binding site on the large subunit. In Specific Aim 3, we will study the process of translocation, whereby the ribosome moves to the next codon. We will attempt to understand the conformational rearrangements that occur during this process, and how EF-G, a GTPase catalyzes the tRNA movement with respect to the ribosome. We will also determine the structure of ribosomal protein S21, which is thought to bridge the two ribosomal subunits. In Specific Aim 4, the global process of translation will be monitored using single-molecule fluorescence. We will develop methods to measure the overall rate of elongation, and how the ribosome is affected by structure in the mRNA. The ribosome is the target of many therapeutic agents. We have extensively studied the aminoglycoside antibiotics. In Specific Aim 5, we will examine the effects of aminoglycosides on ribosomal decoding and translocation; we will measure affinities of the drugs for various states of the ribosome. We will also investigate the global effects of aminoglycoside antibiotics on overall translation. In summary, this proposal attempts to use a combination of biochemical and biophysical methods to unravel the remaining dynamic mysteries of the ribosome, and how these dynamic processes are affected by antibiotics.